Draft genomes announcement of Vietnamese Xanthomonas euvesicatoria strains causing bacterial spot on pepper

Xanthomonas euvesicatoria the primary causal agent of bacterial spot of pepper (BSP), poses a significant global challenge, resulting in severe defoliation and yield losses for pepper growers. We present the whole genome sequences of eight X. euvesicatoria strains associated with BSP in Vietnam. These genomes contribute to representation of pepper production regions in the global sample of X. euvesicatoria genomes, enabling the development of precise global disease management strategies.


INTRODUCTION
In the realm of agricultural sciences, the study of plant diseases and their causal agents holds paramount significance due to their far-reaching impacts on global food security.Xanthomonas euvesicatoria, a bacterium within the Xanthomonadaceae family, is one such pathogen that has necessitated considerable attention for a long time.This bacterium is well-known for causing bacterial spot disease in two economically important crops, tomatoes, and peppers [1][2][3][4].The global significance of this pathogen emphasizes the need for in-depth genomic research to grasp its genetics and evolution, crucial for effective disease control strategies .
Vietnam, known for its diverse agricultural landscape, ranks as the seventh largest pepper producer in the Asia-Pacific region with production of 99 039 tons [5].Approximately 75 % of Vietnam's chillies pepper production is destined for international markets, securing its position as the second largest exporter of dry chillies, contributing over 18.6 % of the global exports in 2021 [6].This production greatly contributes to the country's agricultural exports and strengthens the economic prosperity of Vietnam.Thus, there is a need to address economically damaging diseases such as BSP, particularly when considering the lack of genomic data available for Xanthomonas strains affecting pepper in Vietnam.
In this study, we present a draft genome announcement of X. euvesicatoria isolated from Vietnam.This draft genome announcement serves as a preliminary report on the genomic information for X. euvesicatoria from Vietnam, facilitating in-depth studies on its biology, epidemiology, and regional diversity.Further research using these genomes can provide useful information when developing disease management strategies in Vietnam and beyond.

METHODS
In 2013, symptomatic pepper leaves from eight different locations including five provinces/cities of Vietnam were collected.Sections of individual lesions from a single leaf were macerated in 25 µl of sterile tap water, streaked on Nutrient agar (NA) in a quadrant pattern, and then incubated at 28 °C for 3-5 days to isolate single colonies.Pathogenicity and pepper race determinations in pepper differentials were determined as explained in Subedi et al. [4].Briefly, the pepper cultivar 'Early California Wonder' (ECW) and its near-isogenic lines carrying resistance genes (Bs1, Bs2, Bs3) were infiltrated with bacterial suspensions of 10 8 c.f.u.ml −1 .The infiltrated leaves were observed for hypersensitive or susceptible responses at five time points (12,24,36,48, and 72 hpi).Strains were categorized into races based on responses in the differential lines, with susceptibility confirmed in ECW [7].Strains were also assessed for amylolytic activity by streaking bacterial cells from 24 h cultures grown on NA onto 1.5 % soluble starch-amended NA plates and incubating at 28 °C for 48 h.Amylase activity was determined by observing the presence of a turbid halo around each colony.
Pure cultures of isolated strains preserved at −80 °C were streaked on NA and incubated at 28 °C.Fresh bacterial cultures after 24 h growth were transferred into test tubes containing nutrient broth and incubated overnight at 28 °C on a shaker set at 250 r.p.m.Genomic DNA was extracted from overnight nutrient broth cultures using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) for Gram-negative bacteria.The extracted DNA was sent to Seq Centre (Pittsburgh, PA) and sequenced on the Illumina Next-Seq2000 platform, yielding 151 bp paired-end reads.Illumina DNA Prep kit and IDT 10 bp UDI indices were used to prepare sample libraries.The bcl-convert (v3.9.3), a proprietary Illumina software, was employed for demultiplexing, quality control, and adapter trimming.Raw reads were assembled following the procedures described in Timilsina et al. [8].

RESULTS AND DISCUSSION
All the strains were pathogenic on pepper cultivar ECW and were identified as pepper race P1.The prevalence of race P1 restricts the utilization of Bs1 resistance gene, but there remains a promising opportunity to harness the effectiveness of other commercially available dominant resistant genes, Bs2 and Bs3 for the management of BSP in Vietnam.Prior to the availability of sequencing technology, one of the techniques for differentiation of Xanthomonas species causing bacterial spot in tomato and pepper involved assessing their amylolytic activity.In previous studies X. euvesicatoria strains were either negative or weakly amyloytic [1,7]; however, in another study a few X. eucvesicatoria strains from Mexico and from Ohio in the USA had amylolytic activity [1,16].In this study, all strains from Vietnam were non-amylolytic, which is typical of X. euvesicatoria strains.However, there is an increasing number of reports indicating the presence of amylolytic X. euvesicatoria strains [4,[17][18][19].This finding highlights the need for a more extensive and comprehensive study, incorporating a larger and more varied sample sizes, to better understand the pathogen dynamics.
Genome analysis of eight Vietnamese strains revealed average genome lengths of 5.18 Mbp ranging from 5.12 to 5.24 Mbp, with an average contig count of 88 (range=65 to 95) and an average sequencing coverage of 62 (range=56 to 70).The average N50 of all genomes was 193 424 bp (Table 1) .Whole-genome average nucleotide identity (ANI) analysis confirmed that all sequenced Vietnam strains belonged to X. euvesicatoria, with >99.78 % ANI compared to the X. euvesicatoria type strain ATCC 11633 and <98.54 % ANI compared to the X. perforans type strain DSM_18975 (Fig. 1).The genomic similarity among the sequenced strains varied from 99.81-100 %.Annotation by NCBI predicted 95 RNA genes and 52 tRNA genes in all sequenced strains except VTM17 (RNA genes=94; tRNA genes=51).The number of predicted protein-coding genes ranged from 4189 to 4324 (average=4265).
Despite the relatively limited number of samples and the absence of apparent phenotypic diversity, ANI revealed a degree of genetic variation among X.euvesicatoria strains in Vietnam.For instance, VTM17 stands out from other strains with its lower ANI of 99.8 % setting it apart from the rest, which have ANIs of over 99.9 %.This genetic diversity might be linked to other factors, including the presence of specific pathogenic or virulence-related genes in the strains and the potential antibiotic or bactericide resistance genes.This emphasizes how genetic analysis can reveal subtle differences within the populations, even when visible traits don't provide clear distinctions.Deeper investigations, incorporating genomes from around the world, will provide insights into their genetic relationships with other strains and populations, including variation in effectors.This knowledge can be harnessed for the development of targeted strategies for global disease management.We greatly appreciate the comments/suggestions by all reviewer, which were carefully considered and incorporated into the revised manuscript.The point-by-point responses to all the comments are as follows.

Reviewers' comments and responses to custom questions:
Reviewer 1 Comments to Author:l.37:Please describe whether a single leaf was used for each isolation, or whether multiple leaves were pooled (if known, please state how many), as this is not clear from the text.
Re: Revised as suggested -l.38: Please state length of incubation for obtaining individual colonies (presumably in aerobic conditions?) Re: Revised as suggested -l.47: Please state conditions for growth of isolates for gDNA extraction.
Re: Revised as suggested -l.48:I recognize that a 3rd party sequenced your strain, but you must provide details about Illumina library preparation and sequencing, such as kit name and vendor, with any modifications to manufacturer's protocol.
Re: Revised as suggested -l.49: Please state the number of raw reads obtained.
Re: Revised as suggested.The number of raw reads are included in Table 1.
-l.50: "Trim Galore" -> "TrimGalore"; also please provide a version number for this software, and state parameters used.
Re: Revised as suggested -l.51: Please rephrase (unless it was your intent) as this implies that k-mer coverage of >2.0 was discarded.
Re: Revised as suggested -l.52: Please make it clear which reads are being referred to as "validated reads" -I could not tell.
Re: Revised as suggested -l.52: Please provide a version number for SAMtools Re: Revised as suggested -l.53: Please state a version number for Pilon, and how many rounds of polishing were applied.
Re: Revised as suggested -l.71,Table 1: Please state exact assembled genome length and GC% content for each sequenced isolate.This could be included in table 1.
Re: Revised as suggested.The genome length is presented in base pairs and GC% are also added in Table1.
-Table 1, l.90: Please upload raw reads to SRA and provide the corresponding SRA accessions for each sequencing run/isolate.A BioProject accession is not sufficient for reproducibility as further read data can be added to a BioProject.
Re: Revised as suggested.The raw reads were already deposited in NCBI, and we have also updated the SRA accessions in Table 1.
-ll.80-81:A problem with the phrasing here is that ANI is relative to the reference/comparator genome, and the statement does not include that, as it stands.Table 2 shows the authors' intent (ANI% vs all other sequenced strains), but this does not come across as written.
Re: We revised the sentence; however, we completely do not understand this comment what does the review wants us to address.
In the sentence we have addressed the ANI of all Vietnam strain with reference to Type strains and argue why they belong to X. euvesicatoria.
-For all software, the parameters used (even if they are default) must be listed.If default parameters are routinely used, a blanket statement could be made, e.g."Unless otherwise noted, default parameters were used for all software." Re: Revised as suggested -Please provide the necessary metadata as indicated above.
Re: Revised as suggested -Please provide future revisions of this manuscript formatted in landscape.
Re: Revised as suggested

Reviewer 2 Comments to Author:
The paper is clear, concise, and well-structured.With the exception of the comment beneath about adding an additional sentence or two on the context of the current availability of sequencing data for X. euvesicatoria, the paper has clear reasoning for how these strains will be of use to the wider community for a pathogen that has not been well characterized in isolates from Vietnam previously.The methodology used, both the key analytical stages, and the particular software used for the analyses are both sound.I have one question about the filtering of the assembly as mentioned in the comments below.The results are presented in a clear and easy-to-digest way and all of the deposited data is clearly labeled and easily accessible.
I think a couple of details could be changed slightly to improve the paper as suggested in the comments below.In particular, I think it would be good to give the context of these genomes with what is already available to the community.Looking at the accession for the type strains it looks like LMG27970 is still a relatively fragmented genome, with nearly 1,500 contigs in the assembly.However, there are several complete X. euvesicatoriagenomes available on NCBI.What was the reason for this type strain in particular?The other type strain used only has three contigs.It would be worth including the completeness information with your assemblies for comparison and pointing that your assemblies are comparable/better than type strains in terms of size/ N50 etc. However overall, i'm happy with the paper with a few minor adjustments.
Re: Thank you for looking at the type strains information.We selected this type strain from here https:// lpsn.dsmz.de/.However, we did not realize about the fragmented genome for LMG27970.In the revised version we have replaced the Type strain of Xanthomonas euvesicatoriaLMG27970 with ATCC 11633 with less fragmented genome with 121 contigs.With the new genome of Type strain of Xanthomonas euvesicatoria, our results are consistant.

Comments:
The document orientation is in landscape for most pages rather than portrait, it would be good to edit this and leave only the tables in a landscape format.
Re: Revised as suggested Line 14: Substantial economic losses -if figures are known, it would be good to add a number/statistic here.
Re: Revised as suggested.We do not know the exact figures for the economic losses caused by this disease.Hence, we replace the word substantial with 'severe defoliation and yield losses. ' Line 15: I would say they're not contributing to the representation of all pepper production regions -they are adding the representation of one region.Instead, something along the lines of these genomes gives better representation of the diversity of X. euvesicatoriagenomes in Vietnam, a region that is currently underrepresented in the genomes available for X. euvesicatoriadespite being one of the larger pepper producers in the Asia-Pacific region.
Re: Revised as suggested Line 23: Are there any suitable newer references?
Re: Revised as suggested Line 29: Lack of genomic data for Vietnam.There are 161 X. euvesicatoriagenomes currently submitted in NCBI, including yours.What is the geographic split, are yours the only Vietnamese ones currently?I would add a sentence saying the broad geographical regions covered by the sequencing data currently out there.
Re: Since our focus is mostly on making the genome announcement from Vietnam as these are the first whole genomes of Xanthomonas euvesicatoriafrom Vietnam available in the NCBI database.At this point we do not want to discuss the geographical distribution of all the available 161 Xanthomonas euvesicatoriagenomes in NCBI.
Line 37: Where are the 3 that aren't from 5 cities in Vietnam from?
Re: The rest 3 locations are also from one of 5 cities/provinces (samples were collected from multiple location from some cities/ provinces, that is why its 8 places from 5 cities and provinces.The details of locations are presented in Table1.
Line 51: You say you discard the contigs bigger than 500bp and k-mers higher than 2 for the assembly, should this be < rather than >?It makes sense to exclude the shorter reads and lower coverage regions.
Re: Revised as suggested.We really appreciate for noticing this.This was a typing error and yes, this should be less than.
Line 53: Which version of pilon?
Re: Revised as suggested Line 55: Why these type strains?
Re: As per the definition of type strains which are the first strain of the species identified.These type strains are also the first strain of the Xanthomonas euvesicatoriaand Xanthomonas perforansidentified as type strains.
Line 64: What is the amylolytic activity of the type strains?
Re: Thank you for this comment.We think that this is not important for our study and also, we do not have the physical presence of culture to test their amylolytic activity.
Line 80: I would change the ANI percentage to two significant figures, this makes the difference a bit clearer (like it is in table 2).VTM17 looks a lot more similar to the type strains than the other strains you isolated.
Re: Revised as suggested Line 82: genetic diversity linked to other factors could be checked by Amrfinder or something similar.I am aware this is a genome announcement so discussing this would be out of the scope, but if you ran it and found nothing, it might be worth mentioning.
Re: Thank you for this suggestion, however we feel that this would be out of scope for this study as we would like to stick only to genomes announcement.
Table 1: The species column is redundant because all of the strains have the same value.I would remove this column.It would be useful to put the type strain information in this table for comparison.
Re: We have added the information on type strains hence we decided to keep species column since, there will be two different species (X.euvesicatoriaand X. perforans(Type strain)) Table 2: I would call this a figure rather than a table.I would add in the legend that the type strains are shown in red.
Re: Revised as suggested

Comments:
The paper is clear, concise and well-structured.With the exception of the comment beneath about adding an additional sentence or two on the context of the current availability of sequencing data for X.euvesicatoria, the paper has clear reasoning for how these strains will be of use to the wider community for a pathogen that has not been well characterized in isolates from Vietnam previously.The methodology used, both the key analytical stages, and the particular software used for the analyses are both sound.I have one question about the filtering of the assembly as mentioned in the comments below.The results are presented in a clear and easy-to-digest way and all of the deposited data is clearly labeled and easily accessible.I think a couple of details could be changed slightly to improve the paper as suggested in the comments below.In particular, I think it would be good to give the context of these genomes with what is already available to the community.Looking at the accession for the type strains it looks like LMG27970 is still a relatively fragmented genome, with nearly 1,500 contigs in the assembly.However, there are several complete X.euvesicatoria genomes available on NCBI.What was the reason for this type strain in particular?The other type strain used only has three contigs.It would be worth including the completeness information with your assemblies for comparison and pointing that your assemblies are comparable/better than type strains in terms of size/ N50 etc. However overall, i'm happy with the paper with a few minor adjustments.Comments: The document orientation is in landscape for most pages rather than portrait, it would be good to edit this and leave only the tables in a landscape format.Line 14: Substantial economic losses -if figures are known, it would be good to add a number/statistic here Line 15: I would say they're not contributing to the representation of all pepper production regions -they are adding the representation of one region.Instead, something along the lines of these genomes gives better representation of the diversity of X.euvesicatoria genomes in Vietnam, a region that is currently underrepresented in the genomes available for X.euvesicatoria despite being one of the larger pepper producers in the Asia-Pacific region Line 23: Are there any suitable newer references?Line 29: Lack of genomic data for Vietnam.
There are 161 X.euvesicatoria genomes currently submitted in NCBI, including yours.What is the geographic split, are yours the only Vietnamese ones currently?I would add a sentence saying the broad geographical regions covered by the sequencing data currently out there.Line 37: Where are the 3 that aren't from 5 cities in Vietnam from?Line 51: You say you discard the contigs bigger than 500bp and k-mers higher than 2 for the assembly, should this be ?It makes sense to exclude the shorter reads and lower coverage regions.Line 53: Which version of pilon?Line 55: Why these type strains?Line 64: What is the amylolytic activity of the type strains?Line 80: I would change the ANI percentage to two significant figures, this makes the difference a bit more clear (like it is in table 2).VTM17 looks a lot more similar to the type strains than the other strains you isolated.Line 82: genetic diversity linked to other factors could be checked by Amrfinder or something similar.I am aware this is a genome announcement so discussing this would be out of the scope, but if you ran it and found nothing, it might be worth mentioning.1, l.90: Please upload raw reads to SRA and provide the corresponding SRA accessions for each sequencing run/isolate.A BioProject accession is not sufficient for reproducibility as further read data can be added to a BioProject.-ll.80-81:A problem with the phrasing here is that ANI is relative to the reference/comparator genome, and the statement does not include that, as it stands.
Table 2 shows the authors' intent (ANI% vs all other sequenced strains), but this does not come across as written.-For all software, the parameters used (even if they are default) must be listed.If default parameters are routinely used, a blanket statement could be made, e.g."Unless otherwise noted, default parameters were used for all software."-Please provide the necessary metadata as indicated above -Please provide future revisions of this manuscript formatted in landscape.

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript Good

Table 1 .
Genome statistics of Vietnamese Xanthomonas euvesicatoria strains isolated from pepper along with type strains of Xanthomonas euvesicatoria ATCC 11633 and Xanthomonas perforans DSM-18975

Fig. 1 .
Fig. 1.Pairwise average nucleotide identity and alignment coverage shown in percentage between the sequenced Xanthomonas euvesicatoria strains and with type strains -Xanthomonas euvesicatoria ATCC 11633 and Xanthomonas perforans DSM-18975.The type strains are shown in red.
Thank your for the vised version of your manuscript and for addressing the comments raised by the reviewers.I would like to ask you to make sure that Table1is readible as the cells are in an awkward size and the words are spread over several lines currently, it is hard to read and please change the page format to portrait and only keep landscape for the Table/Figure.
Author response to reviewers to Version 1Dear Editor and reviewers,Thank you very much for your constructive suggestions on our manuscript ID: ACMI-D-23-00221 on 'Draft Genomes Announcement of Vietnamese Xanthomonas euvesicatoriastrains causing Bacterial spot on Pepper.

Table 1 :
The species column is redundant because all of the strains have the same value.I would remove this column.It would be useful to put the type strain information in this table for comparison.Table2: I would call this a figure rather than a table.I would add in the legend that the type strains are shown in red.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.-l.37:Please describe whether a single leaf was used for each isolation, or whether multiple leaves were pooled (if known, please state how many), as this is not clear from the text.-l.38:Please state length of incubation for obtaining individual colonies (presumably in aerobic conditions?) -l.47: Please state conditions for growth of isolates for gDNA extraction.-l.48:I recognize that a 3rd party sequenced your strain but you must provide details about Illumina library preparation and sequencing, such as kit name and vendor, with any modifications to manufacturer's protocol.-l.49:Please state the number of raw reads obtained.-l.50: "Trim Galore" -> "TrimGalore"; also please provide a version number for this software, and state parameters used.-l.51:Pleaserephrase (unless it was your intent) as this implies that k-mer coverage of >2.0 was discarded.-l.52:Please make it clear which reads are being referred to as "validated reads" -I could not tell.-l.52:Please provide a version number for SAMtools -l.53: Please state a version number for Pilon, and how many rounds of polishing were applied.-l.71,Table1:Please state exact assembled genome length and GC% content for each sequenced isolate.This could be included in table 1. -Table